Technical Field
The present disclosure relates to dual-cell model and Caenorhabditis elegans model systems for measuring neuron-to-neuron transmission of protein aggregates, and more particularly to transgenic cell and animal model systems expressing fusion proteins of N-terminus or C-terminus of fluorescent proteins with α-synuclein proteins, methods for measuring continuous cell-to-cell transmission of α-synuclein aggregates using the same, and methods for screening substances for preventing or treating neurodegenerative diseases.
Related Art
Abnormal deposition of α-synuclein aggregates is a pathological feature of Parkinson's disease (PD) (Jellinger, K. A. 2003. Acta Neuropathol 106, 191-201). While a large body of recent studies suggests that transcellular transmission of α-synuclein aggregates drives the progression of PD (Danzer, K. M., et al. 2012 Mol Neurodegener 7, 42), the mechanisms underlying such transmission are not clearly understood. Particularly urgent issues include whether cell-to-cell transmission of aggregates is seeding-dependent, and whether the aggregates disseminate to large cell populations through continuous transmission and the role of other PD-related genes in this process (Lee, H. J. et al., 2014 Nat Rev Neurol).
Genetic and pathological evidence suggests that lysosomal impairment is a major contributor in the pathogenesis of Lewy body diseases (Pan, T. et al., 2008 Brain). The GBA1 gene encodes a lysosomal hydrolase, glucocerebrosidase (GCase), which is deleted in Gaucher disease, the most common lysosomal storage disease. Moreover, mutations in GBA1 are strong genetic risk factors in PD and in dementia with Lewy bodies, although the mechanism by which mutations in GBA1 increase the risk of PD remains unclear (Sidransky, E. et al. 2009 N Engl J Med 361, 1651-1661; Nalls, M. A. et al. 2013 JAMA neurology 70, 727-735). α-synuclein aggregates that are transferred from cell to cell are transported through the endolysosomal pathway and are degraded in lysosomes (Lee, H. J. et al. 2008 Int J Biochem Cell Biol 40, 1835-1849). The present inventors hypothesized that GBA1 deficiency causes lysosomal dysfunction, thereby increasing the efficiency of aggregate transmission.
The present inventors found, in the studies of the mechanism of perpetual transmission of α-synuclein aggregates through continuous cell-to-cell transmission, association of formation and transmission of α-synuclein aggregates with lysosomal function and aging in cells, that bimolecular fluorescence complementation (BiFC) system can facilitate measuring cell-to-cell transmission of α-synuclein aggregates, and developed the present invention.
BiFC is a technique of applying already known complementation of protein fragments to fluorescent proteins, fragmenting the fluorescent proteins into N-terminal fragments and C-terminal fragments and expressing these fragments, respectively, with two proteins between which interaction is to be studied, and analyzing fluorescence of whole fluorescent proteins occurring when the two fragments of the fluorescent proteins are linked to each other, while the two proteins are close to each other for interaction. Hu et al. reported that it is possible to analyze protein-protein interactions in higher animal cells using BiFC (Hu et al., Mol. Cell 2002, 9:789-798). Additionally, in recent years, many results of protein-protein interaction analysis results using BiFC are reported.